The phosphate group serves important functional purposes in biochemistry. It contributes to the structure and function of all major classes of organic biomolecules comprising proteins, lipids, carbohydrates and nucleic acids, along with precursors, combinations and metabolites of these substances. As biomolecules function under aqueous conditions, it is important to have chemical and physical tools and techniques for the detection and manipulation of organic phosphate compounds in their naturally occurring state, i.e., under aqueous conditions.
A gap that has existed in the study of phosphate compounds under aqueous conditions is the ability to specifically and usefully label the phosphate group of such compounds in a single step. Single step labeling means that other functional groups which may be present on the same or other compounds in the sample are not labeled, and that one does not form multiple derivatives of the targeted phosphate group which would make the labeling confusing and therefore not useful.
A great variety of labeling reagents bearing a detectable signal group and a reactive functional group are employed in analytical chemistry, including biochemistry, as has been reviewed (L. J. Kricka, Nonisotopic DNA Probe Techniques, Academic Press, San Diego, 1992). Most used for signal groups are radioisotopes (e.g., .sup.32 P, .sup.33 P, .sup.35 S, .sup.125 I, .sup.3 H, .sup.14 C) , fluorophores (e.g., fluoresceins, rhodamine, tetramethylrhodamine, Texas Red, pyrene, coumarins, dansyl, BODIPY.RTM.s), lumiphores (e.g., luciferin, luminol, isoluminol, lanthanide chelates, acridinium esters), enzymes (e.g., horseradish peroxidase, alkaline phosphatase), haptens (e.g., digoxigenin, biotin, fluorene, dinitrophenyl), coenzymes (e.g., biotin), proteins (e.g., avidin, phycobiliproteins), nucleic acids, plastic particles (e.g., latex), liposomes (e.g., dye or enzyme-filled liposomes), metals (e.g., ferrocene), electrochemiphores (e.g., catechols, sugars), electrophores (e.g., pentafluorobenzyloxyphenyl ketones) and chromophores (e.g., Dabsyl, Malachite Green). The following reactive functional groups, or analogs or activated forms of these groups, have been employed on labeling reagents to form a stable bond to the target functional group on the substance of interest: succinimidyl esters, anhydrides, thiols, maleimides, acyl azides, acyl halides, isocyanates, sulfonyl chlorides, sulfonyl fluorides, hydrazines, amines, alkyl halides, haloacetyls, alcohols, aldehydes, glyoxals, hydrazides, and carboxylic acids (e.g., carbodiimide-activated). But none of these functional groups has been used, or has any ability to be used, to achieve specific labeling of a phosphate compound in a single step under aqueous conditions.
Polynucleotides containing a free phosphate group have been labeled on the phosphate moiety under aqueous conditions by reacting them with ethylenediamine in the presence of a water-soluble carbodiimide, followed by reaction of the residual amino group with an amino-reactive fluorophore (L. E. Morrison et al., Anal. Biochem. 183:231-244, 1989). However, not only does this procedure involve two reaction steps, but each of the reaction steps is nonspecific. Thus, if any contaminating carboxylic acids are present in the first reaction, they will also undergo coupling to the ethylenediamine. Further, if any contaminating amines are present in the second reaction, they will undergo labeling with the amine-reactive fluorophore.
Fluorescein-histamine has been prepared to stain (noncovalently) histamine or histamine blocker sites on mammalian cells (H. R. Petty, U.S. Pat. No. 4,830,961). A polymeric form of this type of reagent also has been introduced (M. E. Osband et al., U.S. Pat. No. 4,474,876). However, the reagents have not been used, or suggested for use, for covalent labeling of phosphate substances, or any kind of covalent labeling.
Mononucleotides have been labeled under aqueous conditions in a single step using a water-soluble carbodiimide with 2-(N-dansyl)-aminoethanol (M. G. Ivanovskaya et al., Nucleosides and Nucleotides 6:913-939, 1987). However, this is not a specific reaction for phosphate compounds, since carboxylic acids will also be labeled under these reaction conditions.
R. Haugland and H. C. Kang (U.S. Pat. No. 4,744,339) have disclosed that a dipyrromethaneboron difluoride dye can be substituted with a chemically reactive group capable of forming a chemical bond with a ligand.